Our goal is to purify several folate requiring enzymes from rabbit liver and to determine their structural properties with respect to multifunctional activities and the formation of enzyme clusters. We have purified two enzymes which show multifunctional catalytic activities. Our goal is to isolate specific domains of these proteins which contain only one of the catalytic activities and to determine the interaction of these domains in the native enzyme. The principal methods to be used are limited proteolysis of the proteins with the isolation of active fragments by HPLC molecular sieve chromatography and the use of differential scanning calorimetric investigations. The formation of enzyme clusters among folate requiring enzymes will include molecular weight studies and kinetic determinations of the "channeling" of the product of one reaction to the active site of the next reaction without total equilibration with the bulk solvent. The enzymes to be studied include formyl-methenyl-methylenetetrahydrofolate synthetase, formiminotransferase-cyclodeaminase, 5-formyltetrahydrofolate cyclodehydrase, and serine hydroxymethyltransferase.